Background: Correct daily skin care is important regarding hand eczema (HE). However, only a few studies have evaluated the level of knowledge among hospital cleaners. Objective: To examine the level of knowledge regarding skin protective behaviour and risk factors with respect to HE in hospital cleaners. Methods: All cleaners working in four hospitals in Denmark were invited to participate in the study, which was based on a questionnaire consisting of 22 questions. Results: 142 of 236 cleaners (response rate = 60.1%) were included. The mean of total correct answers were approximately five out of nine questions. 85.6% of participants evaluated themselves to have a low degree of knowledge of skin care and protection. Based on the number of correct answers, 90% were defined as having low knowledge. Being born or raised outside of Denmark was statistically significantly associated with lower level of knowledge. Risk factors for HE such as, work with wet hands, and use of household cleaning products were only recognized as risk factors by less than 50%. Conclusion: Our study indicates that Danish hospital cleaners have a low degree of knowledge regarding skin care and protection. Cleaning workers born or raised outside of Denmark are in need of special attention.